Method and system for observing a sample under ambient lighting

ABSTRACT

A method for observing a sample is placed between a light source and an image sensor, comprising at least 10000 pixels, the light source emits an illuminating beam, which propagates to the sample, the light beam is emitted in an illumination spectral band (Δλ 11 ) lying above 800 nm, the method comprising the following steps: (a) illuminating the sample with the light source; (b) acquiring an image of the sample (I 0 ) with the image sensor, no image-forming optics being placed between the sample and the image sensor; and (c) the image sensor being configured such that it has a detection spectral band (Δλ 20 ), which blocks wavelengths in the visible spectral band, such that the image may be acquired in ambient light.

TECHNICAL FIELD

The technical field of the invention is related to the observation of asample, in particular a biological sample, with an imaging deviceoperating in ambient light.

PRIOR ART

The observation of samples, and in particular biological samples, bylensless imaging has seen substantial development over the last tenyears. This technique allows a sample to be observed by placing itbetween a light source and an image sensor, without placing anyimage-forming lenses between the sample and the image sensor. Thus, theimage sensor collects an image of a light wave transmitted by thesample, without conjugation between the image sensor and the sample.

Document WO2008090330 for example describes a device allowing biologicalparticles to be observed by lensless imaging. The biological particlesare for example cells. The device allows an interference pattern themorphology of which allows the type of cell to be identified to beassociated with each cell. Lensless imaging would thus appear to be asimple and inexpensive alternative to a conventional microscope. Inaddition, its field of observation is clearly much larger than it ispossible for that of a microscope to be.

In the visible domain, lensless imaging has been applied to examinesamples containing particles, in particular biological particles orcells, for characterization purposes. Examples may be found inWO2017178723, WO2016151248, or WO2016151249. The use of lensless imagingto count particles is described in WO2018115734 or in WO2015166009, orin WO2018060589.

Documents WO2016189257 or EP 319 9941 described the use of lenslessimaging to characterize tissue slides of pathology-slide type.

In the aforementioned documents, the lensless imaging is implementedusing wavelengths in the visible. In order to keep the ambient light atbay, the devices are such that the main components (light source, imagesensor) and the sample are combined in a chamber that is impermeable tolight.

The inventors propose a device that is simple to implement, and thatallows the constraint on isolation of the sensor with respect to ambientlight to be relaxed. A greater ease-of-use results therefrom.

SUMMARY OF THE INVENTION

A first subject of the invention is a method for observing a sample, thesample being placed between a light source and an image sensor,preferably comprising at least 10000 pixels, the light source emittingan illuminating beam, which propagates to the sample, the illuminatingbeam being emitted in an illumination spectral band lying above 800 nm,the method comprising the following steps:

a) illuminating the sample with the light source;

b) acquiring an image of the sample with the image sensor;

the image sensor being configured such that it has a detection spectralband that to blocks wavelengths in a visible spectral band, lying atleast between 400 nm and 750 nm, or at least between 400 nm and 780 nm.

Thus, the image may be acquired in ambient light, the image sensor beingexposed to light in the visible spectral band.

According to one embodiment, no image-forming optics are placed betweenis the sample and the image sensor. According to another embodiment, thedevice comprises an optical system, placed between the sample and theimage sensor, the optical system having an object focal plane and animage focal plane. The device is then such that:

-   -   the object focal plane is offset with respect to a plane in        which the sample lies, by a focusing distance, which is        preferably comprised between 10 μm and 2 mm    -   and/or the image focal plane is offset with respect to the        detection plane, by a focusing distance, which is preferably        comprised between 10 μm and 2 mm.

Whatever the embodiment, the method may comprise any one of thefollowing features, implemented alone or in any technically realizablecombination:

-   -   the detection spectral band is comprised between 800 nm and 1200        nm or between 800 nm and 1000 nm;    -   the illumination spectral band is comprised between 800 nm and        1200 nm or between 800 nm and 1000 nm;    -   the illumination spectral band has a bandwidth narrower than or        equal to 50 nm, and preferably narrower than 20 nm;    -   the detection spectral band has a bandwidth narrower than or        equal to 50 nm, and preferably narrower than 20 nm;    -   the detection spectral band is defined by a high-pass or        band-pass detection filter placed on the image sensor, the        detection filter being configured to block wavelengths in the        visible spectral band; the detection filter may notably be        placed between the image sensor and the sample;    -   the illumination spectral band is defined by an illumination        filter, coupled to the light source; the illumination filter may        notably be placed between the light source and the sample;    -   in step b), the image sensor is exposed to an exposure light        wave; the method may then comprise applying a holographic        reconstruction operator to the image acquired in b), so as to        obtain an image representative of a complex expression of the        exposure light wave. The complex expression may be defined on a        reconstruction surface, for example a reconstruction plane,        lying facing the image sensor, at a nonzero reconstruction        distance from the latter. The reconstruction surface is        preferably a plane in which the sample lies. The application of        the holographic reconstruction operator may be achieved by        implementing an iterative holographic reconstruction algorithm,        so as to to determine a phase of the exposure light wave in the        sample plane or in a detection plane in which the image sensor        lies.

A second subject of the invention is a device for observing a sample,comprising:

-   -   a light source, configured to emit an illuminating beam that        propagates toward the sample, in an illumination spectral band;    -   a pixelated image sensor, comprising at least 10000 pixels, and        configured to acquire an image in a detection spectral band;    -   a holder, arranged to hold the sample between the light source        and the image sensor;        the device being configured such that no image-forming optics        are placed between the image sensor and the sample when the        sample is held on the holder; the device being characterized in        that:    -   the detection spectral band lies above 800 nm;    -   the detection spectral band blocks wavelengths in a visible        spectral band, lying at least between 400 nm and 750 nm, or at        least between 400 nm and 780 nm.

According to one embodiment, no image-forming optics are placed betweenthe sample and the image sensor. According to another embodiment, thedevice comprises an optical system, placed between the sample and theimage sensor, the optical system having an object focal plane and animage focal plane. The device is then such that:

-   -   the object focal plane is offset with respect to a plane in        which the sample lies, by a focusing distance, which is        preferably comprised between 10 μm and 2 mm    -   and/or the focal plane is offset with respect to the detection        plane, by a focusing distance, which is preferably comprised        between 10 μm and 2 mm.

Whatever the embodiment, the method may comprise any one of thefollowing features, implemented alone or in any technically realizablecombination:

-   -   the detection spectral band is comprised between 800 nm and 1200        nm or between 800 nm and 1000 nm;    -   the image sensor is coupled to a detection filter, defining the        detection spectral band;    -   the illumination spectral band is comprised between 800 nm and        1200 nm or between 800 nm and 1000 nm;    -   the illumination spectral band has a bandwidth narrower than or        equal to 50 nm, and preferably narrower than 20 nm;    -   the detection spectral band has a bandwidth narrower than or        equal to 50 nm, and preferably narrower than 20 nm;    -   the light source is a source of laser light;    -   the light source is a light-emitting diode;    -   the light source is coupled to an illumination filter, the        illumination filter defining the illumination spectral band;    -   the device comprises a processing unit configured to apply a        holographic reconstruction operator to the image acquired by the        image sensor, so as to obtain a complex image of an exposure        light wave to which the image sensor is exposed during the        acquisition of the image.

Other advantages and features will become more clearly apparent from thefollowing description of particular embodiments of the invention, whichare given by way of nonlimiting example, and shown in the figures listedbelow.

FIGURES

FIGS. 1A and 1B are examples of devices according to the invention.

FIG. 2 shows the transmission spectral bands of a Bayer filter.

FIGS. 3A and 3B are examples of images acquired using a reference deviceaccording to the prior art and according to the invention, respectively.FIGS. 3C and that 3D are details of regions of interest delineated inFIGS. 3A and 3B, respectively. FIGS. 3E and 3F are profiles obtainedfrom the FIGS. 3C and 3D, respectively, along lines respectively drawnon the latter.

FIG. 4 shows another embodiment of a device.

DESCRIPTION OF PARTICULAR EMBODIMENTS

FIG. 1A shows an example of a device 1 allowing the invention to beimplemented. A light source 11 is configured to emit a light beam 12,called the illuminating beam, which propagates in the direction of asample 10. The illuminating beam reaches the sample by propagating alonga propagation axis Z.

The illuminating beam is emitted in an illumination spectral band Δλ₁₂.The illumination spectral band Δλ₁₂ preferably lies outside of thevisible spectral band. By visible spectral band, what is meant is aspectral band comprised between 400 nm and 750 nm, or between 400 and780 nm. Preferably, the illumination spectral band Δλ₁₂ lies between 750nm or 780 nm and 10 μm, and preferably between 800 nm and 10 μm, andpreferably between 750 nm or even 800 nm and 5 μm, and more preferablybetween 750 nm or even 800 nm and 2 μm, or between 750 nm or even 800 nmand 1200 nm, or between 750 nm or even 800 nm and 1000 nm.

By lies between m and n, m and n representing wavelength values, what ismeant is that more than 80% of the intensity of the emitted light, oreven more than 90% or 95% of the emitted intensity, is comprised betweenm and n. The term lies between m and n does not necessarily mean extendfrom m to n.

The sample 10 is a sample that it is desired to characterize. It notablycomprises a medium 10 _(m) in which particles 10 _(p) bathe. The medium10 _(m) may be a liquid medium. It may comprise a bodily liquid, forexample obtained from blood or urine or lymph or cerebrospinal fluid. Itmay also be a question of a culture medium, comprising nutrimentsallowing microorganisms or cells to develop. By particle, what isnotably meant, non-exhaustively, is:

-   -   a cell, whether it be a question of a cultured cell or a bodily        cell, a blood cell for example;    -   a microorganism, for example a bacterium or a yeast or a        microalgae;    -   a solid particle, for example a microsphere, the microsphere        possibly being functionalized, so as to promote the graft of an        analyte;    -   a particle forming an emulsion in the medium 10 _(m), in        particular a particle that is insoluble in the medium 10 _(m),        an example being a lipid droplet in an aqueous medium.

A particle 10 _(p) may be solid or liquid.

The sample 10 may be a thin slide of biological tissue, such as apathology slide. The thickness of such a slide is of the order of a fewtens of microns.

The sample 10 is, in this example, contained in a fluidic chamber 15.The fluidic chamber 15 is for example a Gene Frame® fluidic chamber ofthickness e=250 μm. The thickness e of the sample 10, along thepropagation axis, typically varies between 10 μm and 1 cm, and ispreferably comprised between 20 μm and 500 μm.

The sample lies in a plane P₁₀, called the sample plane. The sampleplane P₁₀ is preferably perpendicular to the propagation axis Z, orsubstantially perpendicular to the latter. By substantiallyperpendicular, what is meant is perpendicular to within an angulartolerance, for example to within ±10% or ±20%. The sample plane isdefined by the axes X and Y shown in FIGS. 1A and 1B. The sample is heldon a holder 10 s at a distance d from an image sensor 20.

The distance D between the light source 11 and the fluidic chamber 15 ispreferably larger than 1 cm. It is preferably comprised between 2 and 30cm. Advantageously, the light source 11, seen by the sample, may beconsidered to be point-like. This means that its diameter (or itsdiagonal) is preferably smaller than one tenth and better still onehundredth of the distance between the fluidic chamber 15 and the lightsource. In FIG. 1A, the light source is a light-emitting diode. It isgenerally associated with a diaphragm 18, or spatial filter. Theaperture of the diaphragm is typically comprised between 5 μm and 1 mm,and preferably between 50 μm and 500 μm.

The diaphragm may be replaced by an optical fibre, a first end of whichis placed facing the light source 11 and a second end of which is placedfacing the sample 10. The device shown in FIG. 1A also comprises adiffuser 17, placed between the light source 11 and the diaphragm 18.The use of such a diffuser allows constraints on the centeredness of thelight source 11 with respect to the aperture of the diaphragm 18 to berelaxed, as described in EP3221688.

Alternatively, the light source may be a laser source, such as a laserdiode, as shown in FIG. 1B. In this case, it is not useful to associatetherewith a spatial filter or a diffuser.

Preferably, the illumination spectral band 44 ₄₂ has a bandwidthnarrower than 100 nm. By spectral bandwidth what is meant is a fullwidth at half maximum of said spectral band. Preferably, theillumination spectral bandwidth Δλ₁₂ is narrower than 50 nm, or evennarrower than or equal to 20 nm.

The sample 10 is placed between the light source 11 and the image sensor20. The image sensor 20 defines a detection plane P₀, preferably lyingparallel, or substantially parallel to the plane P₁₀ in which the samplelies. The expression substantially parallel means that the two elementsmay not be rigorously parallel, an angular tolerance of a few degrees,of the order of ±20° or ±10° being acceptable.

The image sensor 20 is able to form an image I₀ of the sample 10 in thedetection plane P₀. In the example shown, it is a question of a CCD orCMOS image sensor 20 comprising a matrix array of pixels. The imagesensor comprises a number of pixels preferably higher than 10000, andmore preferably higher than 100000. The detection plane P₀ preferablylies perpendicular to the propagation axis Z. The distance d between thesample 10 and the matrix array of pixels of the image sensor ispreferably comprised between 50 μm and 2 cm, and more preferablycomprised between 100 μm and 2 mm.

The absence of image-forming or magnifying optics between the imagesensor 20 and the sample 10 in this embodiment will be noted. This doesnot prevent focusing micro-lenses potentially being present level witheach pixel of the image sensor 20, said micro-lenses not performing thefunction of magnifying the image acquired by the image sensor, theirfunction being to optimize detection effectiveness. The image sensor 20is configured to form an image in a detection spectral band Δλ₂₀.Advantageously, the detection spectral band does not lie in the visiblespectral band, or if it does does so negligibly. It preferably liesbetween 750 nm or 780 nm and 10 μm, and preferably between 800 nm and 10μm, and more preferably between 750 nm or even 800 nm and 5 μm, and evenmore preferably between 750 nm or even 800 nm and 2 μm, or between 750nm or even 800 nm and 1200 nm, or between 750 nm or even 800 nm and 1000nm. Because it lies outside of the visible spectral band, the detectionspectral band Δλ₂₀ allows images to be acquired when the device 1, andnotably the image sensor 20, is exposed to ambient light, in the visiblespectral band. The detection spectral band is configured such that theimage acquired by the image sensor 20 is not affected, or affectednegligibly, by the ambient light. Thus, the device 1 may be used withoutit being necessary to place it in a chamber that is impermeable tolight. It may be used in ambient light. The ambient-light level in whichthe device is able to operate depends on the fraction of the visiblespectral band detected by the image sensor.

Preferably, the detection spectral band Δλ₂₀ has a bandwidth narrowerthan 100 nm. By spectral bandwidth, what is meant is a full width athalf maximum of said spectral band. Preferably, the width of thedetection spectral band Δλ₂₀ is narrower is than 50 nm, or even narrowerthan or equal to 20 nm.

It will be understood that the detection spectral band Δλ₂₀ and theillumination spectral band Δλ₁₂ overlap at least partially.

The detection spectral band Δλ₂₀ may be defined by the intrinsicproperties of the pixels. The image sensor then comprises pixels able todetect photons solely in the detection spectral band. More simply, thedetection spectral band Δλ₂₀ may be defined by a detection filter 29, ofhigh-pass or band-pass type, placed between the image sensor 20 and thesample 10. Analogously, the illumination spectral band Δλ₁₂ may bedefined by the intrinsic properties of the light source 11. This isnotably the case when the light source is a laser, as shown in FIG. 1B.The illumination spectral band may be defined by an illumination filter19, placed between the light source and the sample. Use of anillumination filter 19 is conventional when the light source 11 is awhite light source or a light-emitting diode.

The image sensor 20 may be an RGB CMOS sensor comprising pixels thedetection spectral band of which is defined by a Bayer filter. Thus, thepixels of the image sensor are sensitive in spectral bands correspondingto the colours red, green and blue of the visible spectral band,respectively. FIG. 2 shows the detection passbands defined by the Bayerfilter. The x-axis corresponds to wavelength, expressed in nm, whereasthe y-axis corresponds to the transmission, i.e. to the percentage oflight flux transmitted. The dotted, dashed and solid curves correspondto the passbands in the blue, green and red, respectively. This type ofcurve is conventional in the field of standard RGB image sensors. It maybe seen that beyond 850 nm, the transmission is equivalent in eachspectral band. Beyond 1000 nm, the transmission decreases. Thus, whenthe image sensor is a standard RGB sensor, it is preferable for thedetection spectral band to be comprised in the interval [750 nm-1100nm], and preferably in the interval [850 nm-1000 nm]. The same goes forthe illumination spectral band. Pixels the transmission of which isuniform, while being sufficient to form an exploitable images, are thenobtained. The image sensor 20 then behaves as a monochromic sensor. Withthis type of image sensor, i.e. one comprising a Bayer filter, thedetection spectral band is defined by a high-pass or band-pass detectionfilter 29 defining the detection passband.

As mentioned in the patent applications cited with respect to the priorart, under the effect of the incident light wave 12, the particles 10_(p) present in the sample may generate a diffracted wave 13, liable toproduce, in the detection plane P₀, interference, in particular with aportion 12′ of the incident light wave 12 transmitted by the sample.Moreover, the sample 10 may absorb some of the incident light wave 12.Thus, the light wave 14, transmitted by the sample, and to which theimage sensor 20 is exposed, which light wave is called the “exposurelight wave”. The exposure light wave 14 may comprise:

-   -   a component 13 resulting from the diffraction of the incident        light wave 12 by is each particle of the sample;    -   a component 12′ resulting from the transmission of the incident        light wave 12 by the sample, some of the latter possibly being        absorbed in the sample.

These components form interference in the detection plane. Thus, theimage I₀ acquired by the image sensor comprises interference patterns(or diffraction patterns), each interference pattern possibly beingassociated with one particle 10 _(p) of the sample.

A processing unit 21, for example a microprocessor, is able to processeach image I₀ acquired by the image sensor 20. In particular, theprocessing unit 21 is a microprocessor connected to a programmablememory 22 in which a sequence of instructions for performing theimage-processing and computing operations described in this descriptionis stored. The processing unit may be coupled to a screen 24 allowingimages acquired by the image sensor 20 or computed by the processor 21to be displayed.

An image I₀ acquired by the image sensor 20, also referred to as ahologram, may be subjected to a reconstruction, called a holographicreconstruction. As described with reference to the prior art, it ispossible to apply, to the image I₀ acquired by the image sensor 20, aholographic propagation operator h, so as to compute a complex amplitudeA(x,y,z) representative of the exposure light wave 14, and to do so forevery point of coordinates (x,y,z) of the space, and more particularlybetween the image sensor 20 and the sample 10. The coordinates (x,y)designate coordinates, called radial coordinates, parallel to thedetection plane P₀. The coordinate z is a coordinate along thepropagation axis Z, expressing a distance between the sample 10 and theimage sensor 20.

The complex amplitude may be obtained using one of the followingexpressions:

A(x,y,z)=I₀(x,y,z)*h* designating the convolution operator, or, andpreferably,A(x,y,z)=√{square root over (I₀(x,y,z))}*h, or even:

${{A( {x,y,z} )} = {\frac{\sqrt{I_{0}( {x,y,z} )}}{\overset{\_}{I_{0}}}*h}},$

I₀ being a mean of the acquired image.

The function of the propagation operator h is to describe thepropagation of light between the image sensor 20 and a point ofcoordinates (x,y,z), located at a distance |z| from the image sensor.The propagation operator is for example the Fresnel-Helmholtz function,such that:

${h( {x,y,z} )} = {\frac{1}{j\; \lambda \; z}e^{j\; 2\pi \frac{z}{\lambda}}{{\exp( {j\; \pi \frac{x^{2} + y^{2}}{\lambda \; z}} )}.}}$

It is then possible to determine a property of the exposure light wave14, for example the modulus M(x,y,z) and/or the phase y (x,y,z), at thedistance |z|, with:

M(x,y,z)=abs[A(x,y,z)]

φ(x,y,z)=arg[A(x,y,z)]

The operators abs and arg respectively designate the modulus andargument.

The distance |z| is a reconstruction distance.

The complex expression A(x,y,z) of the light wave 14 at any point ofcoordinates (x,y,z) of the space, is such that: (x,y,z)=M(x,y,z)e^(j)^(φ) ^((x,y,z)).

The complex expression A is a complex quantity the argument and modulusof which are respectively representative of the phase and intensity ofthe exposure light wave 14.

By implementing holographic reconstruction algorithms, it is possible todetermine the complex expression A in a reconstruction plane. Thereconstruction plane is preferably parallel to the detection plane P₀and/or to the sample plane P₁₀. A complex image A_(Z) of the exposurelight wave 14 in the reconstruction plane is then obtained.Advantageously, the reconstruction plane is the plane P₁₀ in which thesample 10 lies. In order to obtain a holographic reconstruction of goodquality, the image acquired by the image sensor may be subjected to aniterative reconstruction algorithm. Iterative reconstruction algorithmsare for example described in WO2016189257 or in WO2017162985.

It is possible to form images M_(Z) and ϕ_(z) respectively representingthe modulus or the phase of a complex image A_(Z) in a plane P_(Z)located at a distance |z| from the detection plane P₀, withM_(Z)=mod(A_(Z)) and ϕ_(z)=arg(A_(Z)). When the reconstruction planeP_(Z) corresponds to a plane in which the sample lies, the images M_(Z)and ϕ_(z) allow the sample 10 to be observed with a correct spatialresolution.

Trials

A trial was carried out using a reference device and a device accordingto the invention. Each device comprises:

-   -   an infrared LED light source, emitting about a central        wavelength equal to 980 nm, of 20 nm bandwidth (±10 nm on either        side of the central wavelength);    -   an 8-bit IDS UI-1492LE-M CMOS image sensor composed of 3884×2764        square pixels of 1.67 μm side length;    -   a diaphragm defining a 150 μm aperture placed next to the light        source.

The reference device was placed in a dark chamber, forming a chamberthat was impermeable to light. The device according to the inventioncomprises a detection filter 29 placed directly on the image sensor,defining a detection spectral band centred on 980 nm and of spectralwidth equal to 10 nm. Thus, the detection spectral band lay between 975nm and 985 nm. In this example, the device according to the invention isused in daylight.

A sample, containing micron-sized particles in aqueous solution, wasplaced at a distance of 1.5 mm from the image sensor. FIGS. 3A and 3Bare respectively images acquired by the image sensor, with the referencedevice and with the device is according to the invention, respectively.In these figures, zones of interest have been delineated by dashedlines. FIGS. 3C and 3D correspond to the zoomed-in images of the regionsof interest. FIGS. 3E and 3F show intensity profiles produced with eachfigure, along a dashed line. These profiles show that the image qualitywas equivalent with both devices.

according to another embodiment, schematically shown in FIG. 4, animage-forming optical system 16 is placed between the sample and theimage sensor, the image sensor being arranged in a what is called adefocused configuration. The image-forming optic 16 may comprise a lensor an objective. The image-forming optic 16 defines an object focalplane P_(obj) and an image focal plane P_(m). In the defocusedconfiguration:

-   -   the object focal plane P_(obj) is offset from the plane in which        the sample lies by a distance called the defocus distance;    -   and/or the image focal plane is offset from the detection plane        by a distance called the defocus distance.

The defocus distance may be comprised between 5 μm and 5 mm, andpreferably between 10 μm and 2 mm. In the same way as in a lenslessconfiguration, such a configuration allows an image to be obtained inwhich diffracting elements of the sample, particles for example, appearin the form of diffraction patterns, interference occurring between thelight wave emitted by the light source and propagating to the imagesensor and a diffracted wave generated by each diffracting element ofthe sample. In the example of FIG. 4, the object plane P_(obj) iscoincident with the sample plane P₁₀. The image plane P_(m) is offsetwith respect to the detection plane P₀. The features described withreference to the embodiment shown in FIGS. 1A and 1B may be applied tothe defocused configuration.

However, a lensless-imaging configuration is preferred, because of thelarger observation field that it procures.

The invention will possibly be employed to observe samples in the fieldof biology or health, or in other industrial fields, for example foodprocessing and/or environmental inspection.

1-14. (canceled)
 15. A method for observing a sample, the sample beingplaced between a light source and an image sensor, comprising at least10000 pixels, the light source emitting an illuminating beam, whichpropagates to the sample, the illuminating light beam being emitted inan illumination spectral band lying above 800 nm, the method comprising:a) illuminating the sample with the light source; b) acquiring an imageof the sample with the image sensor, no image-forming optics beingplaced between the sample and the image sensor; wherein: the imagesensor is configured such that it has a detection spectral band thatblocks wavelengths in a visible spectral band, lying at least between400 nm and 750 nm, such that the image may be acquired in ambient light;the illumination spectral band has a bandwidth narrower than or equal to100 nm.
 16. The method according to claim 15, wherein the detectionspectral band is comprised between 800 nm and 1200 nm or between 800 nmand 1000 nm.
 17. The method according to claim 15, wherein theillumination spectral band is comprised between 800 nm and 1200 nm orbetween 800 nm and 1000 nm.
 18. The method according to claim 15,wherein: the illumination spectral band has a bandwidth narrower than orequal to 50 nm, and preferably narrower than 20 nm; and/or the detectionspectral band has a bandwidth narrower than or equal to 50 nm, andpreferably narrower than 20 nm.
 19. The method according to claim 15,wherein the detection spectral band is defined by a high-pass orband-pass detection filter placed on the image sensor, the detectionfilter being configured to block wavelengths in the visible spectralband.
 20. The method according to claim 15, wherein the illuminationspectral band is defined by an illumination filter, coupled to the lightsource.
 21. The method according to claim 15, wherein, in b), the imagesensor is exposed to an exposure light wave, the method comprisingapplying a holographic reconstruction operator to the image acquired inb), so as to obtain an image representative of a complex expression ofthe exposure light wave.
 22. A device for observing a sample,comprising: a light source, configured to emit an illuminating beam thatpropagates toward the sample, in an illumination spectral band; apixelated image sensor, comprising at least 10000 pixels, and configuredto acquire an image in a detection spectral band; a holder, arranged tohold the sample between the light source and the image sensor; thedevice being configured such that no image-forming optics are placedbetween the image sensor and the sample when the sample is held on theholder; wherein: the detection spectral band lies above 800 nm; thedetection spectral band blocks wavelengths in a visible spectral band,lying at least between 400 nm and 750 nm; the illumination spectral bandhas a bandwidth narrower than or equal to 100 nm.
 23. The deviceaccording to claim 22, wherein the detection spectral band is comprisedbetween 800 nm and 1200 nm or between 800 nm and 1000 nm.
 24. The deviceaccording to claim 22, wherein the image sensor is coupled to adetection filter, defining the detection spectral band.
 25. The deviceaccording to claim 22, wherein the illumination spectral band iscomprised between 800 nm and 1200 nm or between 800 nm and 1000 nm. 26.The device according to claim 22, wherein: the illumination spectralband has a bandwidth narrower than or equal to 50 nm, and preferablynarrower than 20 nm; and/or the detection spectral band has a bandwidthnarrower than or equal to 50 nm, and preferably narrower than 20 nm. 27.The device according to claim 22, wherein: the light source is a sourceof laser light; or the light source is a light-emitting diode coupled toan illumination filter, the illumination filter defining theillumination spectral band.
 28. The device according to claim 22,comprising a processing unit configured to apply a holographicreconstruction operator to the image acquired by the image sensor, so asto obtain a complex image of an exposure light wave to which the imagesensor is exposed during the acquisition of the image.